Cartilage Matrix Mineralization Research Articles

Engineered human osteoarthritic cartilage organoids

The availability of human cell-based models capturing molecular processes of cartilage degeneration can facilitate development of disease-modifying therapies for osteoarthritis [1], a currently unmet clinical need. Here, by imposing specific inflammatory challenges upon mesenchymal stromal cells at a defined stage of chondrogenesis, we engineered a human organotypic model which recapitulates main OA pathological traits such as chondrocyte hypertrophy, cartilage matrix mineralization, enhanced catabolism and mechanical stiffening. To exemplify the utility of the model, we exposed the engineered OA cartilage organoids to factors known to attenuate pathological features, including IL-1Ra, and carried out mass spectrometry-based proteomics. We identified that IL-1Ra strongly reduced production of the transcription factor CCAAT/enhancer-binding protein beta [2] and demonstrated that inhibition of the C/EBPβ-activating kinases could revert the degradative processes. Human OA cartilage organoids thus represent a relevant tool towards the discovery of new molecular drivers of cartilage degeneration and the assessment of therapeutics targeting associated pathways.

Role of Galectin-3 in Bone Cell Differentiation, Bone Pathophysiology and Vascular Osteogenesis.

Galectin-3 is expressed in various tissues, including the bone, where it is considered a marker of chondrogenic and osteogenic cell lineages. Galectin-3 protein was found to be increased in the differentiated chondrocytes of the metaphyseal plate cartilage, where it favors chondrocyte survival and cartilage matrix mineralization. It was also shown to be highly expressed in differentiating osteoblasts and osteoclasts, in concomitance with expression of osteogenic markers and Runt-related transcription factor 2 and with the appearance of a mature phenotype. Galectin-3 is expressed also by osteocytes, though its function in these cells has not been fully elucidated. The effects of galectin-3 on bone cells were also investigated in galectin-3 null mice, further supporting its role in all stages of bone biology, from development to remodeling. Galectin-3 was also shown to act as a receptor for advanced glycation endproducts, which have been implicated in age-dependent and diabetes-associated bone fragility. Moreover, its regulatory role in inflammatory bone and joint disorders entitles galectin-3 as a possible therapeutic target. Finally, galectin-3 capacity to commit mesenchymal stem cells to the osteoblastic lineage and to favor transdifferentiation of vascular smooth muscle cells into an osteoblast-like phenotype open a new area of interest in bone and vascular pathologies.

Insulin-like growth factor 1 receptor (IGF1R) signaling regulates osterix expression and cartilage matrix mineralization during endochondral ossification

Insulin-like growth factor 1 receptor (IGF1R) signaling is important for bone formation via endochondral ossification. Igf1r deficient mice show proportional dwarfism and alterations in chondrocyte proliferation, hypertrophy and apoptosis within the growth plate. In addition, gene ablation in mouse demonstrated that IGF1R signaling is important for osteoblast mediated bone mineralization. However, the mineralization in the terminal hypertrophic zone of the growth plate is also an essential step in endochondral ossification preceding bone formation. Therefore, we analyzed the influence of IGF1R signaling on this process by using mice with a specific deletion of Igf1r in chondrocytes. Studies in embryonic metatarsal explant cultures showed that mineralization of the terminal hypertrophic zone was strongly reduced when IGF1R signaling was lacking. This decreased mineralization may in part result from the delay in hypertrophic differentiation in the Igf1r deficient metatarsals. However, mineralization was impaired even stronger than hypertrophy, suggesting a mineralization promoting effect of IGF signaling that is independent of hypertrophic differentiation. We found a markedly decreased osterix expression suggesting that osterix is a downstream target of IGF1R in chondrocytes. MMP13 expression was strongly reduced in metatarsals lacking the IGF1R while alkaline phosphatase expression and activity were less affected. We conclude that endogenous IGF1R signaling is important for growth plate matrix remodeling and calcification leading to bone formation and suggest that regulation of osterix expression and its downstream target MMP13 are part of the underlying mechanism.

An activating Fgfr3 mutation affects trabecular bone formation via a paracrine mechanism during growth

The fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the regulation of endochondral ossification. Fgfr3 gain-of-function mutations cause achondroplasia, the most common form of dwarfism, and a spectrum of chondrodysplasias. Despite a significant number of studies on the role of FGFR3 in cartilage, to date, none has investigated the influence of Fgfr3-mediated effects of the growth plate on bone formation. We studied three mouse models, each expressing Fgfr3 mutation either ubiquitously (CMV-Fgfr3(Y367C/+)), in chondrocytes (Col II-Fgfr3(Y367C/+)) or in mature osteoblasts (Col I-Fgfr3(Y367C/+)). Interestingly, we demonstrated that dwarfism with a significant defect in bone formation during growth was only observed in mouse models expressing mutant Fgfr3 in the cartilage. We observed a dramatic reduction in cartilage matrix mineralization and a strong defect of primary spongiosa. Anomalies of primary spongiosa were associated with an increase in osteoclast recruitment and a defect of osteoblasts at the mineralization front. A significant decrease in bone volume, trabecular thickness and number was also observed in the trabecular bone. Interestingly, no anomalies in proliferation and differentiation of primary osteoblasts from CMV-Fgfr3(Y367C/+) mice were observed. Based on these data, we excluded a potential function of Fgfr3 directly on osteoblasts at 3 weeks of age and we obtained evidence that the disorganization of the growth plate is responsible for the anomalies of the trabecular bone during bone formation. Herein, we propose that impaired FGFR3 signaling pathways may affect trabecular bone formation via a paracrine mechanism during growth. These results redefine our understanding of endochondral ossification in FGFR3-related chondrodysplasias.

Exogenous 1,25-Dihydroxyvitamin D3Exerts a Skeletal Anabolic Effect and Improves Mineral Ion Homeostasis in Mice that Are Homozygous for Both the 1α-Hydroxylase and Parathyroid Hormone Null Alleles

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)(2)D(3)-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1alpha-hydroxylase and PTH null alleles, were treated with 1,25(OH)(2)D(3), sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)(2)D(3)-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D(28K), calbindin-D(9K), and Na(+)/Ca(2+) exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)(2)D(3), Exogenous 1,25(OH)(2)D(3) also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)(2)D(3)-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)(2)D(3) used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.

Exogenous PTH-Related Protein and PTH Improve Mineral and Skeletal Status in 25-Hydroxyvitamin D-1α-Hydroxylase and PTH Double Knockout Mice

We examined the effect of NH2-terminal fragments of PTHrP and PTH in young mutant mice deficient in both PTH and 1,25-dihydroxyvitamin D. Both proteins prolonged murine survival by increasing serum calcium, apparently by enhancing renal calcium transporter expression. The dominant effect on the skeleton was an increase in both endochondral bone and appositional formation without increased bone resorption. PTH-related protein (PTHrP) was discovered as a hypercalcemic agent responsible for the syndrome of humeral hypercalcemia of malignancy, and PTH is the major protein hormone regulating calcium homeostasis. Both proteins have skeletal anabolic actions when administered intermittently. We examined effects of exogenous PTHrP(1-86) and PTH(1-34) in double null mutant mice deficient in both PTH and 25-hydroxyvitamin D-1alpha-hydroxylase [1alpha(OH)ase] to determine the action of these proteins in the absence of the two major regulators of calcium and skeletal homeostasis. Mice heterozygous for the PTH null allele and for the 1alpha(OH)ase null allele were mated to generate pups homozygous for both null alleles. PTHrP(1-86) and PTH(1-34) were administered subcutaneously starting 4 days after birth. Serum biochemistry and skeletal radiology, histology, and histomorphometry were performed, and indices of bone formation, resorption, and renal calcium transport were determined by real time RT-PCR, Western blot, and immunohistochemical approaches. In the double mutant mice, which die within 3 weeks after birth with severe hypocalcemia, tetany, and skeletal defects, exogenous PTHrP and PTH enhanced survival of the animals by improving serum calcium. Both proteins increased renal calcium transporter expression and long bone length and augmented growth plate chondrocyte proliferation, differentiation, and cartilage matrix mineralization. Cortical and trabecular bone mass was increased with augmented osteoblast number and activity; however, bone resorption was not increased. PTHrP and PTH reduced hypocalcemia by enhancing renal calcium reabsorption but not by increasing bone resorption. The major skeletal effects of exogenous PTHrP and PTH were to increase bone anabolism.

Regulation of transglutaminase activity in articular chondrocytes through thrombin receptor-mediated factor XIII synthesis.

Transglutaminases are a family of enzymes that catalyze the formation of epsilon-(gamma-glutamyl)lysine isopeptide bonds in proteins, an activity that has been implicated in the pathogenesis of cartilage matrix mineralization in degenerative arthritis. Type II transglutaminase and thrombin-activatable factor XIII have been identified in articular cartilage. Thrombin, a coagulation protease, is found in pathological synovial fluids, and is known to stimulate transglutaminase activity in non-articular tissues. We investigated the effects of thrombin on transglutaminase activity in porcine articular chondrocytes. Direct addition of thrombin to chondrocyte lysates resulted in increased transglutaminase activity due to proteolytic conversion of factor XIII to XIIIa. Thrombin-treated chondrocyte cultures (0.001 to 2.0 U/ml) also showed increased transglutaminase activity. Thrombin treatment of chondrocyte cultures increased transglutaminase activity as early as 15 minutes after addition, an effect that we attributed to factor XIII activation. Additional stimulatory effects of thrombin were observed in cultured chondrocytes at 4 and 24 hours. A thrombin receptor agonist peptide (TRAP) which activates the PAR1 thrombin receptor mimicked these later effects. Thrombin treatment of chondrocyte cultures increased factor XIII mRNA and protein levels, without affecting levels of type II transglutaminase. Thus, thrombin stimulates transglutaminase activity in articular cartilage by directly cleaving factor XIII and by receptor-mediated up-regulation of factor XIII synthesis. Such increases in potential transglutaminase activity may facilitate pathological matrix calcification in degenerative arthritis.

Calpain function in the differentiation of mesenchymal stem cells.

Calpain is a calcium-activated non lysosomal neutral thiol protease (EC 3.4.22.17) present in a wide variety of eukaryotic cells. Calpain is usually present as an inactive form and is activated by calcium ions and phospholipids. The ability of calpain to alter, by limited proteolysis, the activity or function of numerous cytoskeletal proteins, enzymes, and receptors suggests its involvement in various Ca2+-regulated cellular functions. In this review we focus on the differentiation of mesenchymal stem cells, such as the myoblastic, osteoblastic, chondrocytic, and adipocytic lineages, and the biological significance of calpain in its regulation. Calpain has been implicated in the differentiation of myoblasts through the turnover of glycoproteins. In preosteoblastic cells, calpain is important in mediating the proliferative and prodifferentiating effects of parathyroid hormone and bone morphogenetic proteins. For the differentiation of chondrocytes, calpain is involved in cartilage-matrix mineralization. Furthermore, calpain is required for the differentiation of 3T3-L1 preadipocytes into adipocytes, involving the transcriptional activation of the C/EBP alpha gene and the degradation of the cyclin-dependent kinase inhibitor p27 during the mitotic clonal expansion phase of adipocyte differentiation. We summarize these regulatory effects of calpain on the differentiation of mesenchymal stem cells and speculate on the function and location of calpain in the differentiation processes.

Parathyroid hormone is essential for normal fetal bone formation

Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal skeletal development by comparing newborn mice lacking either PTH, PTH-related peptide (PTHrP), or both peptides. PTH-deficient mice were dysmorphic but viable, whereas mice lacking PTHrP died at birth with dyschondroplasia. PTH-deficient mice uniquely demonstrated diminished cartilage matrix mineralization, decreased neovascularization with reduced expression of angiopoietin-1, and reduced metaphyseal osteoblasts and trabecular bone. Compound mutants displayed the combined cartilaginous and osseous defects of both single mutants. These results indicate that coordinated action of both PTH and PTHrP are required to achieve normal fetal skeletal morphogenesis, and they demonstrate an essential function for PTH at the cartilage-bone interface. The effect of PTH on fetal osteoblasts may be relevant to its postnatal anabolic effects on trabecular bone.

Parathyroid hormone is essential for normal fetal bone formation

Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal skeletal development by comparing newborn mice lacking either PTH, PTH-related peptide (PTHrP), or both peptides. PTH-deficient mice were dysmorphic but viable, whereas mice lacking PTHrP died at birth with dyschondroplasia. PTH-deficient mice uniquely demonstrated diminished cartilage matrix mineralization, decreased neovascularization with reduced expression of angiopoietin-1, and reduced metaphyseal osteoblasts and trabecular bone. Compound mutants displayed the combined cartilaginous and osseous defects of both single mutants. These results indicate that coordinated action of both PTH and PTHrP are required to achieve normal fetal skeletal morphogenesis, and they demonstrate an essential function for PTH at the cartilage-bone interface. The effect of PTH on fetal osteoblasts may be relevant to its postnatal anabolic effects on trabecular bone.

The role of ERG (ets related gene) in cartilage development

Objective Based on function and developmental fate, cartilage tissue can be broadly classified into two types: transient (embryonic or growth-plate) cartilage and permanent cartilage. Chondrocytes in transient cartilage undergo terminal differentiation into hypertrophic cells, induce cartilage-matrix mineralization, and eventually disappear and are replaced by bone. On the other hand, chondrocytes in permanent cartilage do not differentiate further, do not become hypertrophic, and persist throughout life at specific sites, including joints and tracheal rings. While many studies have described differences in structure, matrix composition and biological characteristics between permanent and transient cartilage, it is poorly understood how the fates of permanent and transient cartilage are determined. Previous studies demonstrated that chondrocytes isolated from permanent cartilage have the potential to express markers of the mature hypertrophic phenotype once grown in culture, suggesting that cell hypertrophy is an intrinsic property of all chondrocytes and must be actively silenced in permanent cartilage in vivo. These silencing mechanisms, however, are largely unknown. In this paper, we first review nature of chondrocytes in transient and permanent cartilages and then report the cloning and characterization of a novel variant of ets transcription factor chERG, hereafter called C-1-1, which might be involved in regulation of permanent cartilage development. Design For cloning of a novel variant of chERG (C-1-1), we isolated RNA from the cartilaginous femur or tibiotarsus of Day 17 chick embryos and processed it for reverse transcription-polymerase chain reaction (RT-PCR) with the primers from sequences upstream and downstream of the 81 and 72 bp segments alternatively-spliced in mammals. For investigation of function of chERG and C-1-1, we over-expressed chERG or C-1-1 in cultured chick chondrocytes or the developing limb of chick embryo using a retrovirus (RCAS) system, and examined the phenotype changes in the infected chondrocytes or the infected limb elements. Results C-1-1 is an alternative and novel variant lacking the 27 amino acids segment of chERG that has been reported previously. C-1-1 is preferentially expressed in developing articular cartilage, whereas chERG is preferentially expressed in growth plate cartilage. Growth of articular chondrocytes in culture was accompanied by decreasing C-1-1 expression after several passages, while expression of hypertrophic markers increased. Expression of C-1-1 in cultured chondrocytes inhibited cell hypertrophy, alkaline phosphatase activity, and cartilage matrix mineralization. In contrast, over-expression of chERG promoted chondrocyte maturation and mineralization. Conclusion Our data demonstrate for the first time that chERG and C-1-1 play distinct roles in skeletogenesis and may have crucial roles in the development and function of transient and permanent cartilages.

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage.

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage mitrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.

The role of hepatocyte growth factor in growth plate cartilage

To investigate the physiological role of hepatocyte growth factor (HGF) in endochondral bone formation, we examined the expression of HGF and its receptor c-met and the effects of HGF on growth plate chondrocytes. HGF was highly expressed in the prehypertrophic zone and hypertrophic zone in rat costal growth plate cartilage. The expression of HGF increased in rabbit chondrocytes as they matured in culture. Conversely, c-met expression was down regulated along maturation of growth plate chondrocytes. HGF had weak stimulatory effects on DNA and proteoglycan synthesis of growth plate chondrocytes. However, HGF strongly inhibited expression of terminal differentiation-related phenotypes, such as type X collagen and alkaline phosphatase (APase) synthesis and cartilage matrix mineralization. When HGF was removed from the cultures, cells quickly expressed type X collagen and APase. Once chondrocytes differentiated to mature chondrocytes, HGF did not inhibit further differentiation of these cells. These results suggested that HGF is a negative regulator of terminal differentiation of growth plate chondrocytes..

Cytosolic ionized calcium concentration in isolated chondrocytes from each zone of the growth plate.

In isolated chondrocytes from the growth plate, there is an increase in both the cytosolic ionized calcium concentration and in total cell calcium content as the cells approach the mineralization front. The reserve zone chondrocytes have a cytosolic ionized calcium concentration, [Ca2+]i, of 124 +/- 23 nM and a total cell calcium content, CaT, of 12.8 +/- 6.8 nmol/mg cell protein. Proliferative zone chondrocytes have a [Ca2+]i of 172 +/- 29 nM and a CaT of 16.1 +/- 11.8 nmol/mg cell protein. Hypertrophic zone chondrocytes have a [Ca2+]i of 273 +/- 49 nM and a CaT of 35.8 +/- 16 nmol/mg protein. Chondrocytes isolated from the entire growth plate have a [Ca2+]i of 132 +/- 29 nM and a CaT of 13.2 +/- 2.5 nmol/mg cell protein. Chondrocytes isolated from noncalcifying hyaline cartilage have a [Ca2+]i of 85 +/- 10 nM and a CaT of 11.5 +/- 1.4 nmol/mg cell protein. There appears to be a correlation between intracellular calcium accumulation, an increase in the intracellular ionized calcium concentration, and the process of cartilage matrix mineralization.

Effect of diet on longitudinal bone growth and osteochondrosis in swine.

Weanling gilts were fed either a 12% or 16% protein diet for 10 weeks. Animals fed the 12% protein diet had reduced body weights and reduced longitudinal bone growth as measured in the distal radial growth plate. There was no difference in the growth plate widths between the two animal groups, but there was a significant reduction in the daily rate of cell production in the proliferative zone of animals fed the 12% protein diet. No effect of diet on the rate of expansion of the epiphysis at the articular-epiphyseal junction of the distal femur or humerus could be detected. All animals in both groups had morphologic cartilage lesions consistent with early changes associated with osteochondrosis (OCD), and there was no difference in the lesion morphology between the dietary groups. Areas of disorderly endochondral ossification in the radial growth plate were associated with perpendicular growth cartilage infractions. Growth plate lesions were characterized by increased widths of the maturing cartilage zone without increased width of the proliferative zone or an increase in the daily rate of cell production. Focal growth plate lesions developed because of a transitory inhibition of cartilage mineralization and resorption. Disorderly foci of endochondral ossification beneath articular cartilage were characterized by an area of chondrocyte necrosis which prevented normal cartilage matrix mineralization. Lamellae of cartilage necrosis were also present within the reserve zone of the articular cartilage. These were associated with abnormalities of the cartilage canal vessels, and chondrocyte necrosis was considered to precede degenerative changes in articular cartilage matrix.(ABSTRACT TRUNCATED AT 250 WORDS)